Gld2-catalyzed 3' monoadenylation of miRNAs in the hippocampus has no detectable effect on their stability or on animal behavior

Abstract

Gld2, a noncanonical cytoplasmic poly(A) polymerase, interacts with the RNA binding protein CPEB1 to mediate polyadenylation-induced translation in dendrites of cultured hippocampal neurons. Depletion of Gld2 from the hippocampus leads to a deficit in long-term potentiation evoked by theta burst stimulation. At least in mouse liver and human primary fibroblasts, Gld2 also 3' monoadenylates and thereby stabilizes specific miRNAs, which enhance mRNA translational silencing and eventual destruction. These results suggest that Gld2 would be likely to monoadenylate and stabilize miRNAs in the hippocampus, which would produce measurable changes in animal behavior. We now report that using Gld2 knockout mice, there are detectable alterations in specific miRNA monoadenylation in the hippocampus when compared to wild type, but that these modifications produce no detectable effect on miRNA stability. Moreover, we surprisingly find no overt change in animal behavior when comparing Gld2 knockout to wild-type mice. These data indicate that miRNA monoadenylation-mediated stability is cell type-specific and that monoadenylation has no measurable effect on higher cognitive function.

Keywords: Gld2; behavior; hippocampus; miRNA; monoadenylation.

FIGURE 1.

Gld2 controls miRNA monoadenylation. (A) Scatter plot of mean percentages of 3′ monoadenylated counts for 43 miRNAs in six WT and six Gld2 KO hippocampi. (B) Scatter plot on log scale of the steady-state levels (average read counts) of 43 specific hippocampal miRNAs that are differentially 3′ monoadenylated in WT and Gld2 KO mice as identified in panel A. (C) Bar plot representation of panel A showing mean percentages of 43 specific miRNAs that are differentially monoadenylated in WT and Gld2 KO hippocampus (all have a P-value <0.05; Mann-Whitney U-test). (C, inset) Mean percentages of mir-671-5p that are monouridylated in Gld2 KO hippocampus (P-value <0.05).

FIGURE 2.

WT and Gld2 KO mice exhibit similar behaviors. (A) Marble burying test to measure obsessive-compulsive behavior and aversion to new objects (WT = 12 mice; Gld2 KO = 14 mice; P > 0.05, unpaired t-test). All error bars in the figure refer to SEM. (B) Elevated plus maze used to assess anxiety objects (WT = 11 mice; Gld2 KO = 11 mice; P < 0.05, unpaired t-test). (C) Open field test to measure anxiety (WT = 12 mice; Gld2 KO = 12 mice; P < 0.05, unpaired t-test). (D) T-maze assay of spontaneous alternation used to assess working memory (WT = 11 mice; Gld2 KO = 12 mice; P < 0.05, unpaired t-test). (E) Novel object recognition as a memory and object recognition test (WT = 12 mice; Gld2 KO = 14 mice; P < 0.05, unpaired t-test). (F) Morris water maze to investigate spatial learning and memory; left panel depicts acquisition (WT = 12 mice; Gld2 KO = 13 mice; P < 0.05, unpaired t-test) and right panel depicts reversal (WT = 12 mice; Gld2 KO = 13 mice; P < 0.05, unpaired t-test). (G) Morris water maze probe trial after acquisition phase (WT = 12 mice; Gld2 KO = 13 mice; P < 0.05, unpaired t-test). (H) Passive avoidance test used to assess learning and memory (WT = 3 mice; Gld2 KO = 3 mice; P < 0.05, unpaired t-test). (Hab) Habituation.